A monoclonal antibody to the alpha subunit of Gk blocks muscarinic activation of atrial K+ channels.

The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.

A Novel Panel of Serum Biomarkers for MPM Diagnosis.

Exposure to asbestos is the main cause of malignant pleural mesothelioma (MPM), a highly aggressive cancer of the pleura. Since the only tools for early detection are based on radiological tests, some authors focused on serum markers (i.e., mesothelin). The aim of this study was the evaluation of new serum biomarkers to be used individually or in combination, in order to improve the outcome of patients whose disease would be diagnosed at an earlier stage. Serum and plasma were available from 43 subjects previously exposed to asbestos and 27 MPM patients, all being epithelioid type. All the new markers found differentially expressed in MPM and healthy subjects, by proteomic and genomic approaches, have been validated in the serum by the use of specific ELISA. The combined approach, using tools of genomics and proteomics, is found to be highly innovative for this type of disease and led to the identification of new serum markers in the diagnosis of MPM. These results, if confirmed in a larger series, may have a strong impact in this area, because early detection of this cancer in people at high risk could significantly improve the course of the disease and the clinical approach to an individualized therapy.

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

Regulation of agonist binding to A2A adenosine receptors: effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion.

Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A Gs protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and Gs, we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [3H]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [3H]CGS 21680 binding to rat striatal membranes with about equal potency. The nonhydrolyzable analogs, GDP[S] and GTP[S], were equipotent inhibitors and approx. 100-times more potent than GDP and GTP. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demonstrated the presence of two binding sites in rat striatal membranes with mean values for KD of 5.6 and 343 nM and Bmax of 200 and 942 fmol/mg protein. The high-affinity binding site has the characteristics of the A2A receptor. In the presence both of (0.1 mM) GDP[S] and GTP[S], the KD values for the high-affinity site were increased severalfold, whereas the low-affinity site was no longer detected in filtration assays. Dissociation studies revealed monophasic dissociation curves both in the absence and presence of 0.1 mM GDP[S]. However the K-1 value increased in the presence of guanine nucleotide. We also showed that in bovine striatal membranes agonist binding to A2A receptors was modestly modulated by guanine nucleotides, suggesting differences of receptor Gs-protein-coupling a mechanism in different species. Divalent cations often increase agonist binding to different receptors, whereas Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [3H]CGS 21680 binding to the A2A receptor and determined the requirement of these cations to obtain the modulation of binding by guanine nucleotides. We found that millimolar concentrations of divalent cations were required to obtain an effective interaction between the A2A receptor and Gs. The high-affinity binding of [3H]CGS 21680 to the A2A receptor in rat striatal membranes was dependent on the presence of Mg2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of cysteine residues of rat A2a adenosine receptors in agonist binding.

In the present study, we investigated the role of disulfide bridges and sulfhydryl groups in A2a adenosine receptor binding of the agonist 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680). To evaluate the presence of essential disulfide bridges, rat striatal membranes were incubated with [3H]CGS 21680 in the presence of dithiothreitol and binding of the agonist to membranes was measured. The amount of [3H]CGS 21680 which specifically bound, decreased progressively upon pretreatment of membranes with increasing concentrations of dithiothreitol. Pretreatment of rat striatal membranes with 12.5 mM dithiothreitol for 15 min at 25 degrees C resulted in a 2-fold decrease of A2a adenosine receptor affinity for [3H]CGS 21680, and a reduction in the maximal number of binding sites. The presence of agonist or antagonist ligands protected the A2a adenosine receptor sites from the effect of dithiothreitol. We also examined the susceptibility of A2a adenosine receptors to inactivation by the sulfhydryl alkylating reagent, N-ethylmaleimide. When rat striatal membranes were pretreated with N-ethylmaleimide for 30 minutes at 37 degrees C, a decrease in specific [3H]CGS 21680 binding was observed. Pretreatment of membranes with 1 mM N-ethylmaleimide also resulted in a 2-fold reduction of A2a adenosine receptor affinity for [3H]CGS 21680, as well as a slight decrease in the maximal number of binding sites. Neither agonist nor antagonist ligands were effective in protecting the receptor sites from inactivation by N-ethylmaleimide. In contrast, addition of 100 microM guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate were both effective in protecting the receptor sites from inactivation by N-ethylmaleimide. This protective effect was significant but not complete. Our data suggest that disulfide bridges play a role in the structural integrity of the A2a adenosine receptor, furthermore, reduced sulfhydryl groups appear to be important but we do not yet know if they are on the receptor or on the Gs alpha subunit.

2'-C-Methyl analogues of selective adenosine receptor agonists: synthesis and binding studies.

2'-C-Methyl analogues of selective adenosine receptor agonists such as (R)-PIA, CPA, CCPA, NECA, and IB-MECA were synthesized in order to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A1 and A2A receptors in bovine brain membranes and at A3 in rat testis membranes were determined and compared. It was found that the 2'-C-methyl modification resulted in a decrease of the affinity, particularly at A2A and A3 receptors. When such modification was combined with N6-substitutions with groups which induce high potency and selectivity at A1 receptors, the high affinity was retained and the selectivity was increased. Thus, 2-chloro-2'-C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), which displayed a Ki value of 1.8 nM at A1 receptors, was selective for A1 vs A2A and A3 receptors by 2166- and 2777-fold, respectively, resulting in one of the most potent and A1-selective agonists so far known. In functional assay, this compound inhibited forskolin-stimulated adenylyl cyclase activity with an IC50 value of 13.1 nM, acting as a full agonist.

Suc-[Glu9,Ala11,15]-endothelin-1 (8-21), IRL 1620, identifies two populations of ET(B) receptors in guinea-pig bronchus.

The pharmacological properties of endothelin receptors (ETR) were investigated in guinea-pig bronchus by comparing binding and functional results. In binding assays, both the ET(B) agonists, endothelin-3 (ET-3) and N-suc-[Glu9,Ala11,15]ET-1(8-21) (IRL 1620), and the antagonist, N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D- 1-methoxycarbonyltryptophanyl-D-norleucine (BQ 788), showed biphasic inhibition curves of [125I]-endothelin-1 (ET-1) binding to bronchus membranes prepared from intact or epithelium-deprived tissue. IRL 1620 did not completely displace specifically [125I]-ET-1 bound to these tissue preparations. In the presence of the ET(A)-selective antagonist, cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu) (BQ 123, 1 microM), IRL 1620 displacement curves were shallow but a complete inhibition was reached at a concentration of 1 microM. Both curves were better represented by two-site models. In addition, BQ 788 competition curves became monophasic when binding experiments were performed in the presence of 1 microM BQ 123. The non-selective agonist, ET-1, and BQ 123 inhibited [125I]-ET binding to bronchus membranes in dose-dependent fashions with monophasic curves. The contracting activity of IRL 1620 (0.55 nM- 1.6 microM) was tested on multiple-ring bronchial preparations pretreated with peptidase and cyclo-oxygenase inhibitors. BQ 788 shifted IRL1620 concentration-response curves to the right while BQ 123 did not influence bronchial responsiveness. In addition, a potentiation of the maximal response to the agonist was observed in BQ 788 treated bronchial rings. This effect was abolished by tissue pretreatment with Nomega-nitro-L-argininemethylester (L-NAME) or epithelium removal but not by pretreatment with atropine or iberiotoxin. Our results demonstrate that guinea-pig bronchus contains two populations of ET(B) receptors with different affinities for the ET(B)-selective agonist, IRL 1620. One ET(B) receptor population appears to activate bronchial muscle contraction while another on epithelial cells causes muscle relaxation through the release of nitric oxide (NO).

Regulation of agonist binding to rat ET(B) receptors by cations and GTPgammaS.

Endothelins exert their physiological effects through interaction with cell surface receptors that are members of the G-protein-coupled receptor family. The endothelin receptor subtype B (ET(B) receptor) is abundantly expressed in rat cerebellum. Since agonist binding to G-protein-coupled receptors may be modulated by cations and guanine nucleotides, we investigated the effects of cations and guanosine 5'-O-(2-thiotriphosphate) (GTPgammaS) on 125I-endothelin-1 (125I-ET-1) binding to rat cerebellar membranes. Both Na+ and Mg2+-stimulated 125I-ET-1 binding causing an increase in receptor affinity for the agonist. While the effect of the divalent cation was evident at relatively low concentrations (5-10 mM), the stimulatory activity of the monovalent cation appeared at relatively high concentrations (50 mM). Additive activities of 25-50 mM NaCl and 1 mM MgCl2 suggested that monovalent and divalent cations increased receptor affinity for ET-1 by different mechanisms. In the presence of 5 mM MgCl2, 50 mM NaCl caused an additional modest reduction of the Kd value. Whereas 5 mM MgCl2 affected the displacement curves of both ET-3 and suc-[Glu9, Ala11,15]-endothelin-1 (8-21) (IRL 1620), the influence of 50 mM NaCl on these curves was less substantial. All together, these results suggest that modulation of receptor affinity by NaCl depends on the nature of the displacing agonist. In the presence of 5 mM MgCl2 or 50 mM NaCl, a partial regulation of 125I-ET-1 binding by GTPgammaS was detectable, while in the absence of cations no GTPgammaS-dependent inhibition was evident.

[(3)H]ouabain binding to ox brain membranes: Characterization of a high-affinity binding site.

The characteristics of [(3)H]ouabain binding were studied in frontal cerebral cortex and striatum of ox brain. Specific [(3)H]ouabain binding reached equilibrium in approx. 40 min and was stable for at least 90 min. In frontal cortex membranes, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent dissociation constant (K(d)) of 14 +/- 2.0 nM and a maximal number of binding sites (B(max)) of about 44.7 +/- 3.8 pmol/mg protein. A similar monophasic Scatchard plot was found in striatal membranes. The density of binding sites was of the same magnitude in these two cerebral areas, but striatal sites showed a lower binding affinity. The addition of K(+) or of Ca(2+) to the assay also reduced specific binding and the rank order of inhibitory potencies was similar to that previously reported for inhibition of [(3)H]ouabain binding to rat brain. All cardiac glycosides which were so far tested inhibited specific [(3)H]ouabain binding to frontal cortex membranes. Ouabain was the most potent compound tested (IC(50) 18.3 nM) while digoxigenin was the least potent (IC(50) 112.3 nM). Theophylline was a very weak inhibitor of specific binding. These results suggest the presence of high-affinity [(3)H]ouabain binding sites in ox brain membranes, and are related to previous reports for ouabain binding to human and rat brain.

Lack of stereoselectivity of 8-hydroxy-2(di-N-propylamino)tetralin-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in human pre- and post-synaptic brain regions.

The stereoselectivity of the serotonin1A (5-HT1A) receptor compound 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity was investigated in membranes from human 5-HT pre-synaptic (raphe nuclei) and post-synaptic (hippocampus and prefrontal cortex) regions of autopsy brains. After sample incubation with agonists and antagonists, results showed that both the racemic mixture of 8-OH-DPAT or its (+) and (-) enantiomers behaved as full agonists in the tested brain regions. Enantiomer potency (EC50, nM) and efficacy (percentage of maximal inhibition, %) values were similar in all regions under investigation. However, some inter and intra-region variations in racemic 8-OH-DPAT potency and efficacy have been observed. In particular, the potency of racemic 8-OH-DPAT was higher in the prefrontal cortex and raphe nuclei than in the hippocampus, where it was in fact lower than either single enantiomers. Agonist effects were competitively reversed by 5-HT1A antagonists, although once again a different profile was revealed in the hippocampus. The data underscores the lack of stereospecificity of 8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in either pre- or post-synaptic human brain regions. Moreover, such results have significant implication, as they support the notion that human 5-HT1A receptors might vary from one brain region to the other.

Effects of postmortem delay on serotonin and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in rat and human brain tissues.

The reproducibility of serotonin (5-HT) and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity was assessed in membranes, stimulated by forskolin, of rat frontal cortex postmortem as well as of human fronto-cortical, hippocampal and dorsal raphe tissues obtained from autopsy brains. The results revealed that differences between basal and forskolin-stimulated enzyme activities were still significant after 48 h postmortem in rat cortex and in all human brain regions up to 46 h after death. However, a decrease of about 17 and 26% in forskolin-stimulated adenylyl cyclase activity was observed at 24 and 48 h, respectively, in rat cortex. 5-HT and the 5-HT1A receptor agonist, (+)8-hydroxy-2(di-N-propylamino)tetraline (8-OH-DPAT), were able to inhibit forskolin-stimulated adenylyl cyclase activity in a dose-dependent manner for 48 h after death in rat and human brain. In rat cortex, both 5-HT and (+)8-OH-DPAT potencies (EC50, nM) and efficacies (percent of maximum inhibition capacity, %) varied significantly with postmortem delay. Conversely, in human tissues, postmortem delay and subject age did not modify agonist potencies and efficacies. Furthermore, a regionality of 5-HT potency and efficacy was revealed in the human brain. 5-HT was equally potent in cortex and raphe nuclei, while being more potent but less effective in hippocampus. (+)8-OH-DPAT was more active in hippocampus and raphe nuclei than in cortex. (+)8-OH-DPAT behaved as an agonist in all areas, as its efficacy was similar or greater than those obtained with 5-HT. The (+)8-OH-DPAT dose-response curve was completely reversed by 5-HT1A receptor antagonists in rat cortex and all human brain areas. In conclusion, we suggest here that differences between rat and human brain might exist at the level of postmortem degradation of 5-HT-sensitive adenylyl cyclase activity. In human brain, 5-HT1A receptor-mediated inhibition of adenylyl cyclase seems to be reproducible, suggesting that reliable experiments can be carried out on postmortem specimens from patients with neuropsychiatric disorders.

Gender and age-related variation in adenylyl cyclase activity in the human prefrontal cortex, hippocampus and dorsal raphe nuclei.

The influence of gender and age on adenylyl cyclase activity was investigated, through a Dowex-alumina double step chromatographic procedure, in the prefrontal cortex, hippocampus and dorsal raphe nuclei obtained from autopsy cadavers. Results showed that forskolin-stimulated enzyme activity in raphe nuclei was greater in men than in women; a region-dependent rank order of basal, forskolin-induced adenylyl cyclase activity and percentage forskolin-stimulation was observed in women only. Lastly, basal values correlated positively with forskolin-stimulated adenylyl cyclase activity in all areas except the prefrontal cortex of the male subjects. Positive significant correlations were also found between both forskolin-stimulated enzyme activity and percentage forskolin stimulation and aging in the prefrontal cortex. Overall, the findings suggest that sex and/or age-related differences in brain adenylyl cyclase vary from one cerebral region to the other.

Uric acid metabolism in two patients with coexistent Down's syndrome and gout.

Two patients with coexistent Down's syndrome and gout are described. Although increased serum urate levels are frequently reported in Down's syndrome, only a few such patients have been described with concomitant gout. In our patients no significant alterations of the purine salvage pathway were found, and the turnover parameters of uric acid, determined by means of a 14C-labeled uric acid study, were consistent with the metabolic findings observed in normoexcretor gouty patients.

A structure-activity study of a C-terminal endothelin analogue.

We report a structure-activity study of an endothelin (ET) analogue, obtained by introduction of a non-aminoacidic portion on the C-terminal ET pentapeptide. The peptidic moiety was modified with systematic replacement of each residue by alanine (Ala scan); further modifications were performed at the C-terminus. The biological activity was analyzed at both ET(A) and ET(B) receptor subtypes, showing that the two C-terminal residues (Ile-Trp) are very important for the activity. On the contrary, the aminoacidic central portion of the molecule appears to be much more tolerant toward modifications.

Isolation of an urate-binding protein by affinity chromatography.

This paper describes an affinity chromatography procedure to purify an urate binding protein from human serum. The specific ligand was 8-amino-2,6-dihydroxypurine bound to Sepharose through the amino group. The specific elution was obtained with an uric acid or allopurinol solution. Electrophoretic analysis of the eluted protein shows a single sharp band with an α2-globulin mobility. Molecular weight, determined by gel filtration, is approximately 70,000 daltons.

Synthesis and structure-activity relationship studies of new endothelin pseudopeptide analogues containing alkyl spacers.

We replaced the Asp18-Ile19 dipeptide of the C-terminal ET analogue Ph-Ph-CH2-O-N=CH-CO-Phe-Asp-Ile-Ile-Trp-OH by alkyl spacers of various lengths to investigate the role of the aminoacidic central portion of the molecule and to define the N-terminal and C-terminal pharmacophoric regions of this analogue. The side-chains of the central dipeptide have been shown to be irrelevant for the binding of the molecule to the receptor, but the distance between the two postulated sites of interaction of the ligand with the ETB receptor appears to be fundamental.

Superoxide dismutase-like activity of 1,2,3-triazole derivatives.

This paper reports the synthesis of some sulfurated 1,2,3-triazole derivatives and the biological evaluation, for the superoxide dismutase-like activity, of these new compounds together with several 1,2,3-triazoles previously described by us. All the compounds possess a SOD-like activity and some of them showed an activity equivalent to or higher than those of penicillamine or thiopronin, which are well known therapeutic drugs in the treatment of rheumatoid arthritis.

Toward the rational development of peptidomimetic analogs of the C-terminal endothelin hexapeptide: development of a theoretical model.

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.

Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, ß-arrestin translocation and chemotaxis assays.

BACKGROUND AND PURPOSE: Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells. EXPERIMENTAL APPROACH: We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through ß-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter ß-arrestin translocation. KEY RESULTS: There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses. CONCLUSIONS AND IMPLICATIONS: Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of ß-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.

Modulation of PAR(1) signalling by benzimidazole compounds.

BACKGROUND AND PURPOSE: Recently, a small molecule (Q94) was reported to selectively block PAR(1) /Gα(q) interaction and signalling. Here, we describe the pharmacological properties of Q94 and two analogues that share its benzimidazole scaffold (Q109, Q89). Q109 presents a modest variation from Q94 in the substituent group at the 2-position, while Q89 has quite different groups at the 1- and 2-positions. EXPERIMENTAL APPROACH: Using human microvascular endothelial cells, we examined intracellular Ca(2+) mobilization and inositol 1,4,5-trisphosphate accumulation as well as isoprenaline- or forskolin-stimulated cAMP production in response to thrombin. KEY RESULTS: Q89 (10 µM) produced a leftward shift in the thrombin-mediated intracellular Ca(2+) mobilization concentration-response curve while having no effect on the E(max) . Both Q94 (10 µM) and Q109 (10 µM) reduced intracellular Ca(2+) mobilization, leading to a decrease in E(max) and an increase in EC(50) values. Experiments utilizing receptor-specific activating peptides confirmed that Q94 and Q109 were selective for PAR(1) as they did not alter the Ca(2+) response mediated by a PAR(2) activating peptide. Consistent with our Ca(2+) results, micromolar concentrations of either Q94 or Q109 significantly reduced thrombin-induced inositol 1,4,5-trisphosphate production. Neither Q94 nor Q109 diminished the inhibitory effects of thrombin on cAMP production, indicating they inhibit signalling selectively through the G(q) pathway. Our results also suggest the 1,2-disubstituted benzimidazole derivatives act as 'allosteric agonists' of PAR(1) . CONCLUSIONS AND IMPLICATIONS: The Q94 and Q109 benzimidazole derivatives represent a novel scaffold for the development of new PAR(1) inhibitors and provide a starting point to develop dual signalling pathway-selective positive/negative modulators of PAR(1) .